Vitamin D exerts therapeutic effects on bronchopulmonary dysplasia (BPD), but its underlying mechanisms remain unclear. The present study was designed to investigate the effects of vitamin D on hyperoxia-induced BPD and elucidate the underlying mechanisms. Neonatal rats were exposed to either room air (control) or 75% O2 (hyperoxia) and intraperitoneally injected with vitamin D3. After 14 days, a histopathological examination was performed in the lungs of rats. Serum 25-hydroxyvitamin D (25OHD) was measured by liquid chromatography-tandom mass spectrometry (LC-MS)/MS. Interleukin 1 beta (IL-1β) and interferon gamma (IFN-γ) were measured by specific enzyme-linked immunosorbent assays. The messenger RNA and protein levels of vitamin D receptor (VDR), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), and hypoxia-inducible factor 1α (HIF-1α) were determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblot analysis, respectively. Treatment with vitamin D3 increased serum 25OHD and upregulated VDR in lung tissues with or without hyperoxia. In addition, treatment with vitamin D3 attenuated alveolar simplification, increased VEGF and VEGFR2, and protected alveolar simplification induced by hyperoxia. Furthermore, treatment with vitamin D3 resulted in a decrease of IL-1β and IFN-γ and an increase of HIF-1α in lung tissues under hyperoxia conditions. Vitamin D exerts protective effects on hyperoxia-induced BPD in neonatal rats by regulating vitamin D-VDR signaling pathways. © 2021 Wiley Periodicals LLC.